Plasmid for expressing phosphorylated recombinant proteins in a bacterial system

ABSTRACT

The present invention provides a plasmid containing a promoter sequence, a nucleotide sequence encoding an exogenous protein, and a nucleotide sequence encoding an enzyme capable of modifying the exogenous protein. In a specific embodiment of the invention the encoded exogenous protein is human β-casein and the encoded enzyme is a human kinase capable of phosphorylating recombinant β-casein in a bacterial system. Phosphorylated recombinant human β-casein having 3 or more phosphate groups and synthesized using the plasmid of the invention is shown to have the same bioactivity as native human β-casein.

This application is a continuation-in-part of U.S. Ser. No. 08/395,239filed on Feb. 27, 1995.

TECHNICAL FIELD

This invention relates to a novel method for producing modifiedrecombinant proteins in a bacterial system. The method comprisespreparing a single vector having a nucleotide sequence encoding anexogenous protein and an enzyme capable of modifying the protein invivo, and expressing the vector in the host cell to produce a modifiedprotein. An aspect of the invention relates to a single vectorcontaining a promoter, followed by a protein encoding sequence, followedby an enzyme encoding sequence. Data are presented that show that themodified protein has the same bioactivity as the native human protein.

BACKGROUND OF THE INVENTION

It is generally recognized that human milk is the best nutritionalsource for human infants. Human milk is not only an ideal source ofnutrients for the developing infant, but also contains bothimmunoglobulins and non-immunological factors that protect the infantfrom infection by various organisms. Human milk is also easily digestedby the infant and is less likely to cause allergic reactions than isinfant formula based on bovine milk.

Human milk differs from bovine milk as well as the milk of othermammalian species in various ways. Overall protein content and the kindsof protein differ between human and bovine milk. Four major bovinecaseins have been identified. Bovine milk contains 2 α-caseins plus β-and κ-casein, but human milk contains only β- and κ-casein.Additionally, the amino acid sequences of human milk protein differ fromthat of other mammalian milk proteins.

Efforts have been made to develop infant milk formulas that have some ofthe advantageous properties of human milk and avoid the disadvantagesassociated with bovine milk based infant formulas such as allergicreactions and incomplete digestion by the infant. An intuitivelydesirable method to achieve this is to add to the formula some of theknown constituents of human milk, including human milk proteins in theirnative form. The human caseins, which differ in amino acid sequence fromtheir bovine and other mammalian counterparts, represent importantsubstances which, if added in their native form to infant formula, wouldserve to enhance the nutritional value of the formula and reduce theinherent disadvantages of non-human milk proteins.

In addition to being a source of amino acids necessary for the synthesisof proteins required for the growth and development of infants, humanmilk is recognized as containing proteins, including casein, that haveother important biological functions. β-casein is one of the mostabundant milk proteins synthesized in the mammary gland. Afterpost-translational modification in the Golgi apparatus, it is excretedas large calcium-dependent aggregates called micelles. β-casein is not asingle entity, but is a heterogeneous group of phosphoproteins secretedduring lactation in response to lactogenic hormones. The primarystructure of human β-casein was determined by Greenberg et al.(Journalof Bioloclical Chemistry 259:5132-5138, 1984). It was shown to be aphosphorylated protein with phosphorylation sites at specific seryl andthreonyl residues located near the amino terminus. Comparison of humanand bovine β-caseins showed 47% identity. The sequence of human κ-caseinwas determined by Brignon et al. (Federation of European BiologicalSocieties Letters 188:48-54, 1985). Whereas β-casein is phosphorylated,κ-casein is glycosylated.

Several biological effects have been ascribed to human milk caseinincluding: (1) enhancement of calcium absorption; (2) inhibition ofangiotensin I-converting enzyme; (3) opioid agonism; (4) andimmunostimulating and immunomodulating effects.

Human casein consists largely (>80%) of the β-form with a smaller amountin the κ-form (Greenberg et al., 1984). Native β-casein is a 25 kDaprotein.

In human milk, β-casein molecules show variable degrees ofpost-translational phosphorylation ranging from zero to five phosphategroups per polypeptide chain (Greenberg et al., 1984; Hansson et al.,Protein Expression and Purification 4:373-381, 1993). Phosphate groupsin the native protein are attached to serine and threonine residueslocated near the amino terminus (Greenberg et al., 1984).

Expression of exogenous genes in bacterial cells provides a usefulmethod for producing recombinant eukaryotic proteins. However, bacteria,such as E. coli, are not capable of producing the post-translationalmodifications required by many eukaryotic proteins as they do notpossess the endogenous enzymes necessary to do so. Therefore, eukaryoticproteins produced in E. coli lack the specific post-translationalmodifications which may occur within the eukaryotic cell, such asglycosylation, phosphorylation, acetylation, or amidation.

Prior to the development of appropriate cloning techniques, thephosphorylation of purified proteins by a kinase was done in vitro usingchemical reagents. This process requires the protein substrate and thekinase enzyme to be purified and this is not efficient or cost-effectivefor commercial purposes. The in vitro process is also inefficient whenit is desired to scale-up for commercialization. There is, therefore, aneed to develop a method for genetically engineering microorganisms tophosphorylate a protein in vivo.

Canadian Patent Application No. 2,083,521 to Pawson et al. teaches amethod of producing phosphorylated exogenous protein in host cells. Themethod of Pawson et al. requires two vectors to be introduced into abacterial cell. One vector has a nucleotide sequence encoding anexogenous protein that is capable of being phosphorylated by thecatalytic domain of a protein kinase. The other vector has a nucleotidesequence encoding the protein kinase catalytic domain. Both vectors areintroduced into E. coli and production of the exogenous protein and theprotein kinase catalytic domain is induced so that the exogenous proteinis phosphorylated. The bacterial cells are then lysed and the exogenousphosphorylated protein is isolated using standard isolation techniques.

CA No. 2,083,521 does not suggest or disclose the method of the instantinvention. The present inventors use a single vector expressing both thesubstrate and the kinase enzyme. The method of Pawson et al. requiresthe use of two vectors. The expression system disclosed herein resultsin specific phosphorylation of the exogenous protein as determined byantibody to phosphoserine, while the expression system of Pawson et al.results in non-specific phosphorylation of both host proteins andexogenous protein. This would adversely affect the growth of hostbacteria in scale-up efforts for industrial applications. The presentinvention, unlike that of Pawson et al., provides for high levelproduction of a phosphorylated, recombinant protein suitable forcommercial production.

Simcox et al., Strategies in molecular biology 7 (3):68-69 (1994)constructed two E. coli strains that harbor a tyrosine kinase plasmid.These TK (tyrosine kinase) strains can be used for generatingphosphorylated proteins when transformed with a plasmid containingsequences encoding a phosphorylation target domain or protein. Both E.coli strains carry an inducible tyrosine kinase gene. One strain, TKB1,is useful for expressing genes whose expression is directed by the T7promoter. The system developed by Simcox et al. differs from the presentinvention in that it requires two constructs, i.e., a tyrosinekinase-containing plasmid and a plasmid vector containing a geneencoding a protein or domain to be phosphorylated.

In order to better understand the structure and function of humanβ-casein and to permit studies of factors that affect regulation of itssynthesis and secretion, cDNA for this protein was cloned and sequenced(Lannerdal et al., Federation of European Biological Societies Letters269:153-156,1990), and human milk β-casein was produced in Escherichiacoli and Saccharomyces cerevisiae (Hansson et al., 1993). Hansson et al.demonstrated that recombinant human β-casein was expressed in the yeast,S. cerevisiae, using the pYES 2.0 vector (Invitrogen Corp., San Diego,Calif.). Production levels were estimated to be approximately 10% of theproduction found in E. coli. However, recombinant β-casein obtained fromS. cerevisiae, a eukaryotic cell that has endogenous enzymes capable ofphosphorylating proteins, was phosphorylated, but the protein producedby E. coli, a prokaryotic cell that lacks the ability in its nativestate to phosphorylate, was non-phosphorylated. Subsequently, it wasshown that recombinant human casein kinase II (rhCKII) produced in andpurified from E. coli can phosphorylate protein substrates in vitro (Shiet al., Proceeding of the National Academy of Sciences, USA91:2767-2771, 1994). One specific embodiment of the present inventionuses a nucleotide sequence encoding a recombinant human casein kinase IIin a single construct with nucleotide sequence encoding β-casein totransform E. coli and produce phosphorylated β-casein. None of the priorart suggests or discloses a single vector containing a promoter followedby a nucleotide sequence encoding a protein followed by a nucleotidesequence encoding a kinase as is disclosed in the present invention.

SUMMARY OF THE INVENTION

There is disclosed herein a method for producing a modified recombinantprotein in a host cell comprising preparing a single vector encodingboth an exogenous protein and an enzyme capable of modifying theexogenous protein. Representative of exogenous proteins capable of beingmodified through the process of the present invention include but arenot limited to human caseins, including β-casein, cell receptorproteins, fatty acylated proteins including palmitoylated proteins,mammalian muscle proteins, the gag polyproteins of retroviruses, andmammalian proteins targeted by retroviral src kinases. Transmembraneglycoproteins that acquire covalent palmitate after synthesis includethe insulin, β₂ -adrenergic and transferrin receptors. Proteins thatfunction as cell surface receptors, tyrosine and serine/threoninekinases, their substrates, a phosphatase, G-proteins, and Ca²⁺ are knownto be fatty acylated. Representative of enzymes useful in the presentinvention because of their capacity to transfer functional groups tospecific exogenous proteins in a host cell, include but are not limitedto kinases, such as tyrosine kinases or casein kinase, transferases,such as mammalian and yeast palmitoyl transferases, and kinases codedfor by the src gene of retroviruses. Representative of promoters usefulin the present invention include inducible promoters such as T7, λP_(L),λP_(R), and Tac and constitutive promoters such as bla and spa.Representative of host cells capable of being transformed and thenexpressing the modified proteins, include but are not limited to thebacterial cells E. coli K-12 and E. coli B, Bacillus species,Lactobacillus species, and Streptococcus species and eukaryotic cellssuch as yeast cells or mammalian.

An exogenous protein is one that originates outside the organism that isproducing it. The term is sometimes used in the relevant DNA cloningliterature also to refer to the recombinant protein produced by thetransformed recipient organism. Alternatively, an exogenous proteinproduced using DNA cloning techniques may be referred to as arecombinant protein. The terms will be used interchangeably herein sincethe distinction is frequently not made in the literature. However, indiscussing the disclosed invention the word "recombinant" will be usedto refer to the protein produced by the transformed organism, and"exogenous" will be used when referring to the native, non-recombinantprotein or nucleotide sequence encoding the protein.

What is disclosed herein is a method for producing a modifiedrecombinant protein in a host cell comprising the steps of preparing asingle vector having a promoter sequence, an exogenous protein sequence,and a nucleotide sequence encoding an enzyme capable of modifying theexogenous protein; transforming the host cell with the vector;expressing the vector in the host cell whereby the produced enzymemodifies the produced recombinant protein; and isolating the produced,modified recombinant protein. Also disclosed herein in a more specificembodiment of the invention is a method for producing a phosphorylatedrecombinant protein in a host cell comprising the steps of preparing asingle vector having a promoter sequence followed by a nucleotidesequence encoding an exogenous protein capable of being phosphorylatedby a protein kinase, followed by a nucleotide sequence encoding aprotein kinase capable of phosphorylating the exogenous protein;transforming the host cell with the vector; expressing the vector in thehost cell whereby the produced protein kinase phosphorylates theproduced recombinant protein; and isolating the phosphorylated protein.

More particularly the present inventors have developed a novel methodfor producing a modified recombinant human protein in bacterialexpression systems wherein the resulting recombinant human protein hasutility for the inhibition of attachment of H. influenzae to human cellsand in the prevention and treatment of otitis media in human infants.Using a combination of two human casein kinase encoding sequences,expressing respectively the alpha and beta subunits of the kinase, theydemonstrated the in vivo production of recombinant phosphorylated humanβ-casein in E. coli. The sequence coding for human casein kinase II wasplaced in tandem with the sequence coding for β-casein with the resultthat a significant portion of the recombinant β-casein produced in E.coli was phosphorylated as in human milk. The method of the presentinvention can also be used for in vivo specific glycosylation,amidation, or acetylation of recombinant proteins in transformed hostcells or for the transfer of fatty acids to appropriate recombinantprotein substrates in transformed host cells.

In a specific embodiment of the invention, a nucleotide sequenceencoding a human casein kinase II (hCKII βα) is co-expressed in a singleconstruct with a nucleotide sequence encoding a human β-casein in abacterial expression system to achieve efficient in vivo phosphorylationof the appropriate serine and threonine residues of recombinant humanβ-casein. Experiments in which a nucleotide sequence encoding hCKII βαand a nucleotide sequence encoding human β-casein were co-expressed inE. coli using a single inducible expression vector demonstrated theability of recombinant hCKII βα to phosphorylate recombinant β-casein invivo. This was an unexpected, non-obvious result requiringexperimentation and inventiveness. As was demonstrated by negativeresults obtained in early, control experiments the disclosed inventionshowed unexpected results. The method of the present invention producesuseful and beneficial results which will permit the addition ofbeneficial human proteins to nutritional and pharmaceutical products.

Phosphorylated β-casein produced using the method of the invention isdemonstrated to have the same bioactivity as native human β-casein asshown by its ability to inhibit adhesion of H. influenzae to humanpharyngeal cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows physical maps of expression vectors pS637 and pRJB-6constructed for inducible intracellular expression in E. coli. 191 basepairs were removed from pS637 to produce PRJB-6.

FIG. 2 shows physical maps of expression vectors pRJB-6 and pRJB-9 andillustrates how pRJB-6 was cut and ligated to CKII βα to form pRJB-9.

FIG. 3 shows physical maps of expression vectors pS637 and pRJB-7 andshows how pS637 was cut and ligated to CKII βα to form pRJB-7. pRJB-7has T7 promoters in front of both the 62 -casein and casein kinasegenes.

FIG. 4 shows the physical map of expression vector pS750, constructedfor inducible expression and to mediate production of intracellularlylocalized protein in E. coli.

FIG. 5 shows SDS-PAGE of Met-β-casein produced in E. coli BL21 strainsand stained with Coomassie Brilliant Blue using the vectors pS750 andpET-11d-CKII βα. The codon for methionine (Met) was placed in front ofthe β-casein encoding sequence in the construction of plasmid pS750because in E. coli and other bacteria the synthesis of their proteinsbegins with the amino acid methionine. This enables the ribosome torecognize the starting point for growth of a polypeptide chain.Production of intracellular recombinant β-casein is possible only whenMet is inserted before the encoding sequence for the protein to beproduced. Lane 1: molecular weight marker (Bio-Rad prestained, relativemolecular weights 106, 80, 49.5, 32.5, 27.5, 18.5 kDa); lane 2:non-phosphorylated recombinant β-casein; lane 3: 5P-β-casein; lane 4:pS750 induced with IPTG in BL21(DE3); lane 5: pS750/pET-11d-CKII βαinduced with IPTG in BL21(DE3); lane 6:pS750 induced with IPTG inBL21(DE3)pLysS; lane 7: pS750/pET-11d-CKII βα induced with IPTG inBL21(DE3)pLysS; lane 8: pS750 induced with IPTG in BL21(DE3)pLysE; lane9: pS750/pET-11d-CKII βα induced with IPTG in BL21(DE3)pLysE cells; lane10: native β-casein with five attached phosphate groups (5P-β-casein).The arrow indicates the β-casein band.

FIG. 6 shows SDS-PAGE of Met-β-casein produced in E. coli BL21 strainsstained with Ethyl Stains-All using the vectors pS750 and pET-11d-CKIIβα. Lane 1: native β-casein with five attached phosphate groups(5P-β-casein); lane 2: pS750/pET-11d-CKII βα induced with IPTG inBL21(DE3)pLysE cells; lane 3: pS750 induced with IPTG in BL21(DE3)pLysE;lane 4: pS750/pET-11d-CKII βα induced with IPTG in BL21(DE3)pLysS; lane5:pS750 induced with IPTG in BL21(DE3)pLysS; lane 6: pS750/pET-11d-CKIIβα induced with IPTG in BL21(DE3); lane 7:pS750 induced with IPTG inBL21(DE3); lane 8: 5P-β-casein; lane 9: non-phosphorylated recombinantβ-casein; lane 10: molecular weight marker (Bio-Rad prestained, relativemolecular weights 106, 80, 49.5, 32.5, 27.5, 18.5 kDa). The arrowindicates the phosphorylated β-casein band, which is seen as a greenband in the original photographs.

FIG. 7 shows SDS-PAGE of Met-β-casein produced in E. coliHMS174(DE3)pLysS stained with Ethyl Stains-All using the vectors pS750and pET-11d-CKII. Lane 1: molecular weight marker (Bio Rad prestained);lane 2:pS750 uninduced; lane 3: pS750 induced with IPTG; lane 4:pS750/pET-11d-CKII βα uninduced; lane 5: pS750/pET-11d-CKII βα inducedwith IPTG; lane 6: pET-11d-CKII βα uninduced; lane 7: pET-11d-CKII βαinduced with IPTG; lane 8: native 5P-β-casein; lane 9: recombinantβ-casein; lane 10: molecular weight marker (Bio-Rad prestained, relativemolecular weights 106, 80, 49.5, 32.5, 27.5, 18.5 kDa). The arrowindicates the phosphorylated β-casein band, which is seen as a greenband in the original photographs.

FIG. 8 shows a Western immunoblot analysis using antibody to humanβ-casein. Lane 1: molecular weight marker (Gibco BRL, relative molecularweights 43.1, 29.2, 18.8, 16.5, 6.4 kDa); lane 2:50 ng native humanβ-casein; lane 3: uninduced HMS174(DE3)pLysS(pRJB-7); lane 4: inducedHMS174(DE3)pLysS(pRJB-7); lane 5: uninducedHMS174(DE3)pLysS(pET-11d-CKII βα); lane 6: inducedHMS174(DE3)pLysS(pET-11d-CKII βα); lane 7: uninducedHMS174(DE3)pLysS(pRJB-9); lane 8: induced HMS174(DE3)pLysS(pRJB-9).

FIG. 9 shows a Western immunoblot analysis with antibody tophosphoserine. Lane 1: low molecular weight marker (Gibco BRL, relativemolecular weights 44, 28.7, 18.5, 14.7, 5.8, 2.g kDa); lane 2:1 μgnative human β-casein; lane 3: 2 μg native human β-casein; lane 5:induced HMS174(DE3)pLysS(pET-11d-CKII βα); lane 6: inducedHMS174(DE3)pLysS(pRJB-9); lane 7: induced HMS174(DE3)pLysS(pRJB-7); lane8: induced HMS174(DE3)pLysS(pS637); lane 10: 1 μg recombinant humanβ-casein; lane 11: 2 μg recombinant human β-casein.

FIG. 10 shows an immunoblot analysis using antibody to human β-casein.Lane 1: molecular weight marker (Gibco BRL, relative molecular weights44, 28.9, 18.5, 14.7, 5.8 kDa); lane 2: native human β-casein; lane 3:induced HMS174(DE3)pLysS(pRJB-9); lane 4: inducedHMS174(DE3)pLysS(pS637); lane 5: induced HMS174(DE3)pLysS(pET-11d-CKIIβα); lane 6: recombinant human β-casein.

FIG. 11 shows an immunoblot analysis using antibody to phosphoserine.Lane 1: molecular weight marker (Gibco BRL, relative molecular weights44, 28.9, 18.5, 14.7, 5.8, 2.9 kDa); lane 2:1 μg native human β-casein;lane 3: 500 ng native human β-casein; lane 4: inducedHMS174(DE3)pLysS(pRJB-9); lane 5: induced HMS174(DE3)pLysS(pS637); lane6: induced HMS174(DE3)pLysS(pET-11d-CKII βα); lane 7:1 μg recombinanthuman β-casein; lane 8:500 ng recombinant human β-casein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing a modifiedrecombinant protein in a host cell. In a more specific embodiment theinvention relates to a method for producing a phosphorylated humanprotein in a bacterial cell. The method comprises the steps of preparinga single vector having both a nucleotide sequence encoding an exogenousprotein that is capable of being phosphorylated by a protein kinase anda nucleotide sequence encoding an appropriate protein kinase, expressingthe vector in a host cell whereby the produced kinase phosphorylates theproduced exogenous protein, and isolating the phosphorylated recombinantprotein. The present inventors have made the unexpected discovery thatplacing the nucleotide sequence encoding the protein to bephosphorylated and the nucleotide sequence encoding the kinase in tandemin a single construct with a promoter results in high level and specificphosphorylation while eliminating the negative features associated withmultiple vectors such as the need for antibiotic resistance genes to beused as markers. Use of the single construct system facilitates scalingup the procedure for industrial use. It is contemplated that the methodof the invention will be useful in any host cell system that is capableof expressing the exogenous protein. Suitable host cells include bothprokaryotes such as bacteria and eukaryotes such as yeast and animalcells.

In the preferred embodiment of the present invention the host cell is E.coli. Nucleotide sequences encoding β-casein, in several differentexpression formats, were evaluated for expression of recombinant humanβ-casein in an E. coli strain. After a series of experiments, it wasdetermined that recombinant human β-casein was efficientlyphosphorylated when sequences encoding human β-casein were placed in asingle construct with sequences encoding human casein kinase CKII βα.Efficiency of phosphorylation was not compromised when both genes wereplaced in tandem in one plasmid when compared with experimental systemsin which sequences encoding the kinase and the β-casein were placed intwo separate vectors.

Materials and Methods

The following materials and methods were used in the investigationsdescribed in Examples 1 to 5. Additional materials and/or methods aredescribed for individual experiments when required. Materials andmethods used in Example 6 are separately described.

Plasmids

Plasmid construct pS637 shown in FIG. 1 is identical to pS26,constructed and described in Hansson et al., (1993), which is hereinincorporated by reference, except that it encodes an additional aminoacid, glutamine (Gln), at position 19. The original expression vector,pS26, was modified to create pS637 which produces a recombinant β-caseinprotein identical to the most abundant variant found in humanpopulations.

The construct pS637 was prepared for co-expression with the nucleotidesequence encoding casein kinase II (Shi et al., 1994), which is herebyincorporated by reference, by placing the nucleotide sequence encodingCKII βα, which codes for two casein kinase subunits, β and α, as acassette, downstream from the nucleotide sequence encoding β-casein. Athree-cistron tandem expression vector pET-11d-CKII βα is a plasmidcontaining CKII βα that was generated by Shi et al.(1994). First, pS637was cut at two sites downstream of the β-casein encoding sequence andreligated. A plasmid, pRJB-6, shown in FIG. 1, was isolated which hadlost 191 bases between the two cut sites. The kinase CKII βα wasprepared for insertion into pRJB-6. After insertion the resultingconstruct was designated pRJB-9, which is shown in FIG. 2. pRJB-9 is asingle construct designed to mediate production of phosphorylatedβ-casein. pS637 was also modified to construct the plasmids pS750 andpRJB-7 which will be described in further detail below.

Host Cells

In the specific embodiment of the invention described below the hostorganism transformed by the described vectors was E. coli. Otherrepresentative organisms that could be used with the method of theinvention include Bacillus, Lactobacillus, and Streptococcus species.

Promoter

In the specific embodiment of the invention described below the T7promoter was used. Other representative promoters that could be usedwith the method of the invention include the inducible promoters λP_(L)and λP_(R) and Tac and the constitutive promoters bla and spa.

Construction of Plasmids for Bacterial Expression: Detailed Methods

Expression vector pS637

Expression vector pS637 differs from pS26, described in Hansson et al.(1993) as it contains a nucleotide triplet encoding the glutamine (Gln)amino acid residue at position 19 of the β-casein encoding sequence.This nucleotide sequence was isolated from a human cDNA variant that ismore commonly found in human populations than is the sequence of pS26.Two synthetic oligonucleotides were synthesized for polymerase chainreaction (PCR) amplification. The synthetic oligonucleotides provideconvenient restriction sites and incorporated codons for amino acidsused preferentially by bacteria. The two oligonucleotides weredesignated SYM4174 (Seq. ID NO: 1) and SYM4175 (Seq. ID NO: 2) and havethe following sequences:

    __________________________________________________________________________    SYM4174                                                                            5'-CGCTGCAGCATATGCGTGAAACCATCGAATC-3'                                    SYM4175                                                                            5'-CGGGATCCTGGTCCTGGTCCTCGTGTTTAACTTTTTCAACTTTCTGTTTGTATTCGGTGATCGATT         C-3'                                                                     __________________________________________________________________________

PCR amplification was performed as described in Ausubel et al., (eds.)Current Protocols in Molecular Biology, Vol.2, Supp.16, 15.0.3-15.1.17(1991) and the amplified fragment was digested with PstI and AvaII togenerate an 85 bp fragment. Plasmid pS21, described in Hansson et al.(1993) was digested with EcoRV and AccI and a 328 bp fragment wasisolated by gel electrophoresis. The isolated fragment was purified fromthe agarose gel by electroelution and digested with AvaII. This resultedin a 197 bp AvaII/AccI fragment which was isolated. The 85 bp PstI/AvaIIdigested PCR-amplified fragment and the 197 bp AvaII/AccI were ligatedinto PstI/AccI digested pS25, a plasmid described in Hansson et al. Theresulting plasmid construct was sequenced and designated pS636. A 644 bpNdeI and BamHI restriction fragment was isolated from pS636 andintroduced into NdeI/BamHI digested vector pS26, a plasmid described inHansson et al. The resulting expression vector was designated pS637.

Expression vector pRJB-9

The pET-11d-CKII βα plasmid comprising the CKII βα encoding sequencesgenerated by Shi et al. (1994) was prepared for co-expression withrecombinant β-casein. First, 191 base pairs (bp) were removed from pS637by cutting two EcoR1 sites downstream from the β-casein encodingsequence and religating pS637. A plasmid, pRJB-6 (FIG. 1), was isolated,which had lost the 191 bp between the two sites and had retained asingle EcoRV site located 132 bases away from the 3' end of the β-caseinencoding sequence. The plasmid pET-11d-CKII βα, containing the CKII βαencoding sequence, was cut with ClaI and the site was filled in withKlenow enzyme (Stratagene, Calif.) to create blunt ends. The filled in,ClaI cut CKII encoding sequence was inserted into pRJB-6, downstreamfrom the β-casein encoding sequence, and the resulting construct wasdesignated pRJB-9 and is shown in FIG. 2.

Expression vector pRJB-7

The construct pS637 was prepared for co-expression of recombinantβ-casein and the CKII βα kinase by placing the CKII βα encoding sequenceimmediately after the β-casein encoding sequence. The CKII βα encodingsequence was placed as a BglII/BamH I fragment into the BamH I site ofpS637 and designated pRJB-7. This fragment contained the T7 promoterfrom its original vector, pET-11D-CKII βα. Thus, as shown in FIG. 3,pRjB-7 contains two T7 promoters, one before the β-casein encodingsequence and one before the CKII βα encoding sequence.

Expression vector pS750

To change the selective marker from ampicillin resistance to kanamycinresistance, the plasmid pS637 was digested with PvuI and treated with T4DNA polymerase to generate blunt ends. The linearized vector wasisolated and ligated with a HincII kanamycin resistance genblock(Pharmacia, Uppsala, Sweden). The resulting expression vector wasdesignated pS750 (FIG. 4).

Expression vector for recombinant human casein kinase II

The expression vector pET-11d-CKII βα (Shi et al., 1994) was provided byDr. C. Walsh of the Harvard Medical School, Boston, Mass.

Expression experiments were carried out as described by Studier et al.(Methods in Enzymology 185:60-89, 1990). Bacteria were grown in LuriaBroth (LB medium) containing 50 μg/ml carbenicillin for pET-11d-CKII βα,the plasmid that contains a gene conferring resistance to carbenicillin,and 50 μg/ml kanamycin for the vector pS750, a plasmid containing a geneconferring resistance to kanamycin. The medium was supplemented with 30μg/ml chloramphenicol when the strains containing the pLys plasmids,which confer resistance to chloramphenicol, were used. For induction ofthe T7 expression system, the cultures were grown to a density ofapproximately OD₆₀₀ =0.5, and then 0.4 mM isopropylβ-D-thiogalactopyranoside (IPTG) was added. The cells were harvestedabout 90 minutes after induction.

Electrophoresis and Detection of Recombinant β-Casein

Cells were pelleted by centrifugation and the pellet from 1 ml ofculture was dissolved in 100 μl of sample buffer, which contains Tris,glycerol, SDS, dithiothreotol (DTT), and bromophenol blue. The proteinswere separated by SDS-PAGE as described in Laemmli (Nature 227:680-685,1970). Gradient gels were cast and run in the discontinuous buffersystem in a Protean (Bio-Rad, Richmond, Calif.) electrophoresis unit.Gels were stained as described in Laemmli. Immunoblotting was performedaccording to the specifications of the manufacturer (Bio-Rad).

Procedure for isolation of modified protein

The modified protein can be isolated by any standard procedure known tothose skilled in the art. Representative of such standard procedures isthe following:

Cells are harvested and ruptured by standard mechanical or chemicalprocedures. Cells are then suspended in buffer, homogenized andcentrifuged and the supernatant is discarded. The resulting insolublepellet is resuspended and the supernatant is discarded. This results ina washed insoluble pellet that is suspended in 50 mM Tris and 6M Urea atpH 8.2 and homogenized. β-casein supernatant I is removed resulting inan insoluble extract that is again suspended in 50 mM Tris and 6M Ureaat pH 8.2 and homogenized. β-casein supernatant II is removed andsupernatants I and II are pooled. The remaining insoluble extract isdiscarded. The pooled supernatants are diluted 1:1 with 50 mM Tris andpH 8.2 and treated with 3M Urea to extract β-casein. The final β-caseinsolution is obtained by dialyzing the Urea extract of β-casein against50 mM ethanolamine and 100 mM NaCl at pH 9.5, centrifuging, and dilutingin 50 mM ethanolamine, 100 mM NaCl at pH 9.5 to a protein concentrationof 5 mg/ml. The pellet is discarded.

EXAMPLES

Examples 1 and 2 are provided to form a basis for the claimed invention,but are not part of the invention being claimed. The experimentsdescribed in Examples 1 and 2 show that production of recombinantβ-casein is not adversely affected when bacteria are co-transformed withtwo vectors containing respectively a nucleotide sequence encodingβ-casein and a nucleotide sequence encoding a casein kinase. They alsodemonstrate that recombinant phosphorylated β-casein can be producedusing these two vectors in a bacterial system.

Example 3 demonstrates that the precise structure of the single plasmidwas neither obvious nor expected, but that its construction requiredinventiveness and experimentation. Example 4 describes a system in whicha single construct, containing a promoter and both the nucleotidesequence coding for the protein to be transcribed and phosphorylated andthe nucleotide sequence coding for the kinase, was used to transform abacterial strain. In Example 4, production of recombinant phosphorylatedβ-casein using a single plasmid was demonstrated. A single constructsystem for expression of extracellularly localized recombinantphosphorylated β-casein that is identical to human native β-casein isdescribed in Example 5. Examples 4 and 5 are within the scope of thepresently claimed invention. Example 6 shows a comparison of sixphosphoforms of native human and recombinant human β-caseins made underthe direction of the plasmid of the invention in their ability toinhibit adhesion of the bacterium H. influenzae to human pharyngealcells.

Example 1

Production of β-casein in E. coli B: Phosphorylation of intracellularlylocalized recombinant Met-β-casein: BL21(DE3) strains

To analyze the ability of recombinant human CKII (rhCKII) tophosphorylate recombinant β-casein in vivo in a bacterial expressionsystem, experiments were performed in E. coli using two inducibleexpression vectors. The expression vector pS750 was transformed alone orin combination with expression vector pET-11d-CKII βα into the T7 hoststrains BL21(DE3), BL21(DE3)pLysS, and BL21(DE3)pLysE. DE3 is a DNAfragment derived from a lambda phage containing a lacl repressor, alacUV5 promoter which is inducible by isopropylβ-D-thiogalactopyranoside (IPTG), and a gene for T7 RNA polymerase. Inthe presence of the inducer, 17 RNA polymerase is produced resulting intranscription of the exogenous genes. Plasmid pLysS confers resistanceto chloramphenicol and has little effect on growth rate and productionof foreign protein. It contains a T7 lysozyme that increases stabilityof plasmids in E. coli and permits the cells to be lysed by freezing andthawing.

Results as seen in FIG. 5 indicate that high levels of recombinant humanMet-β-casein were produced in E. coli and that the amount produced wasnot influenced by co-production of recombinant human CKII βα. Afterelectrophoretic separation of the proteins and phosphate staining, CKIIβα is seen to have phosphorylated recombinant human Met-β-casein invivo. This is shown in FIG. 6 and demonstrates the ability to producephosphorylated β-casein in a bacterial system using two vectors. Thisexample is not within the scope of the claims and is provided to assistthe examiner in understanding the inventive nature of the inventiondescribed in detail in Example 4.

Example 2

Production of β-casein in E. coli K-12: Phosphorylation ofintracellularly localized recombinant Met-β-casein: HMS174(DE3)strains

E. coli K-12 strains HMS174(DE3), HMS174(DE3)pLysS, and HMS174(DE3)pLysE were evaluated as hosts for production of recombinanthuman Met-62 -casein and were transformed with pS750. The most efficientproduction was achieved with HMS174(DE3)pLysS. Co-expression experimentsusing pS750 and pET-11d-CKII βα showed strong induction of recombinanthuman Met-β-casein production, which was independent of the presence ofpET-11d-CKII βα. Phosphate staining (FIG. 7) showed efficientphosphorylation of Met-β-casein when co-produced in vivo withrecombinant human CKII. This example, as was also the case for Example 1is not within the scope of the claims, and is also provided to assistthe examiner in understanding the inventive nature of the inventiondescribed in Example 4. A two plasmid system is inherently lessdesirable than the single plasmid system of the present invention aseach of the plasmids must contain an antibiotic marker so that itspresence in the host cells can be monitored during the fermentationprocess. This necessitates the use of two antibiotics in the growthmedium and retards bacterial growth.

Example 3

Production of human β-casein E. coli K-12: Construct pRJB-7 containingboth a β-casein encoding sequence and CKII βα encoding sequences: T7promoter in front of β-casein encoding sequence; T7 promoter in front ofCKII βα encoding sequences

The construct pRJB-7, containing the β-casein and the CKII βα genes eachpreceded by a T7 promoter, was transformed into E. coli K-12 hostHMS174(DE3)LysS. The transformation and induction procedures followedwere those of the Novagen pET system manual as described in Example 4.

Western Blot Analysis

Separation and transfer, blocking and antibody procedures are describedin Example 4. FIG. 8 shows an immunoblot in which production of β-caseinby E. coli HMS174(DE3)LysS cells containing four different constructs iscompared. Lysates from both induced and uninduced cell cultures areanalyzed. Cells contain pET-11d-CKII βα (plasmid with CKII β and αencoding sequences), pRJB-9 (hybrid construct with both β-casein andCKII βα encoding sequences and T7 promoter in front of β-casein encodingsequence only), or pRJB-7 (hybrid construct with both β-casein and CKIIβα encoding sequences and T7 promoters in front of both β-casein andCKII βα encoding sequences). Transformation of the bacteria with pRJB-7resulted in severe reduction of bacterial growth. E. coliHMS174(DE3)LysS had approximately twice the doubling time as did thesame strain transformed with pRjB-9, the construct with only one T7promoter. The Western blot shown in FIG. 8 shows reduced production ofrecombinant β-casein by induced cells containing pRJB7 when comparedwith cells containing pRJB-9. This is seen by comparing lane 4 (inducedpRJB-7) with lane 8 (induced pRJB-9). Although both pRJB-7 and pRJB-9are derived from pS637, only pRJB-9 produced amounts of β-caseinequivalent to the parent construct. The presence of an additional T7promoter before the CKII genes in the hybrid construct had the effect ofboth reducing cell growth and consequently reducing recombinant proteinproduction.

FIG. 9 shows a Western blot analysis in which the lysates were developedwith phosphoserine antibody to detect phosphorylated protein. Induced E.coli HMS174(DE3)LysS cells containing pET-11d-CKII βα, pRJB-9 (hybridconstruct with one T7 promoter), pRJB-7 (hybrid construct with two T7promoters), or pS637 (contains β-casein encoding sequence but not CKIIβα encoding sequence) were compared for production of phosphorylatedrecombinant β-casein. Phosphorylated β-casein was produced only in cellscontaining pRJB-9 (lane 6). No phosphorylated protein was detected inlane 7, which contains the lysate of cells containing pRjB-7.

Failure to detect phosphorylated protein in the construct with two T7promoters indicates that both inventiveness and experimentation wererequired in order to develop the single construct system disclosedherein for expressing an appropriately modified recombinant protein inmicroorganisms. Although the experiment with two T7 promoters in asingle construct containing the nucleotide sequence encoding a proteinand the nucleotide sequence encoding a kinase gave a negative result,under different experimental conditions the use of more than onepromoter sequence should not be excluded. Situations where it would befavorable to use two different promoters remain within the scope of thepresent invention.

Example 4

Production of human β-casein in E. coli K-12: Construct pRJB-9containing both β-casein encoding sequence and CKII βα encodingsequences

The present invention uses a single construct expressing both theinformation for transferring functional groups to specific sites and theprotein to be modified. In a specific embodiment of this invention thetransferred functional group is phosphate. The transfer is accomplishedby a kinase that is demonstrated to mediate phosphorylation of specificsites on recombinant human β-casein in vivo. This invention demonstratesthat not only can human β-casein be specifically phosphorylated in vivoby E. coli, but that a single-construct with a promoter located beforethe sequence encoding β-casein and having the advantages of asingle-construct system can successfully mediate this function.

Transformation into E. coli K-12 HMS174(DE3)pLysS

The construct pRJB-9, containing the β-casein and CKII βα genes, wastransformed into E. coli K-12 host HMS174(DE3)LysS. The transformationprocedure followed was that of the Novagen pET system manual (4th ed.,TB No.55, June, 1994).

Induction of Expression

E. coli HMS174(DE3)LysS host cells containing plasmids pRJB-9 (FIG. 2),pS637 (FIG. 1), or pET-11d-CKII βα (Shi et al, 1994) were grown at 30°C. to a density of OD₆₀₀ 0.5-0.6. Culture samples were taken before and6 hours after adding 1 mM of the inducer IPTG. Cells from two 1 mlaliquots were pelleted by centrifugation in a microcentrifuge. Cellswere resuspended in sample loading buffer for gel electrophoresis afterwhich 500 μl of the supernatants from each aliquot were collected. Thespent culture medium was concentrated in a Microcon 10 spin filter(Amicon) for 35 minutes at 10,000×G. The retentate was collected afterspinning for 3 minutes at 1,000×G and an equal amount of sample bufferat double concentration was added.

Western Blot Analysis

Cell lysates were separated on SDS-Polyacrylamide pre-cast Gel(Integrated Separations System) with a 10-20% gradient and transferredto an Immobilon-P membrane (Millipore, Bedford, Mass.) with a semi-dryblotter. Gels were electroblotted at a constant current (0.8 mA/cm²) for45 minutes onto Immobilon PVDF filters (Millipore) using a Trans-Blot SDTransfer Cell (Bio-Rad) The transfer buffer contained 48 mM Tris, 39 mMglycine, 1.3 mM SDS (sodium dodecyl sulfate) and 20% methanol. Prior totransfer, the filter was soaked first in methanol and then in transferbuffer. For Western blot analysis, the membrane was blocked in 3% bovineserum albumin and 0.2% Tween in TBS (25 mM Tris, 0.154M NaCl pH 7.4).Primary antibody to β-casein and alkaline phosphatase goat anti-rabbitantibody, the secondary antibody, were diluted 1:8000 in the blockingbuffer. An additional antibody was used to detect phosphoserines.Blocking and antibody reactions were done at 25°-26° C. in 2% gelatincontaining amplification grade porcine skin (U.S. Biochemicals) in TBSfor 2 hours. The blot was then rinsed with TBS for 30 minutes. Primaryantibody, mouse monoclonal anti-phosphoserine (Sigma) was diluted 1:200or 1:100 in the 2% gelatin blocker and incubated for two hours. The blotwas rinsed twice in TBS for 5 minutes. The secondary antibody, goatanti-mouse alkaline phosphatase (Sigma), was diluted 1:4,000 in thegelatin blocker, incubated for one hour, and rinsed as before in TBS.Nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate were usedas substrate for color development.

FIG. 10 shows an immunoblot in which production of β-casein by E. coliK-12 HMS174(DE3)LysS cells containing three different constructs iscompared. Cells contain pS637 (plasmid with β-casein encoding sequence),pET-11d-CKII βα (plasmid with CKII β and α encoding sequences), orpRJB-9 (hybrid construct with both β-casein and CKII βα encodingsequences). Comparison of lanes 3 and 4 shows that the hybrid construct,pRJB-9, is producing equivalent amounts of β-casein to pS637, from whichit was derived and which does not contain the CKII βα encodingsequences. Both pRjB-9 and pS637 produced between 400-500 mg/L ofβ-casein in this host cell. This experiment shows that placing theβ-casein encoding sequence in tandem with the encoding sequence for CKIIβα does not significantly change production of β-casein.

FIG. 11 shows a Western blot analysis in which the lysates weredeveloped with phosphoserine antibody to detect phosphorylated protein.Increased quantities of native human β-casein and non-phosphorylatedrecombinant β-casein were tested in addition to the lysates of FIG. 8.No phosphorylation of bacterial proteins is seen in lane 6, whichcontains the lysate from the CKII βα plasmid, showing thatphosphorylation is specific. The cell lysate in lane 4, containingpRJB-9 with the β-casein and CKII βα encoding sequences in tandem, showsa strong band cross-reacting with the antibody. The band of lane 4 hasthe same molecular weight as native human milk β-casein byelectrophoretic analysis as seen in lanes 2 and 3. There was nocross-reactivity to recombinant, non-phosphorylated human β-casein,either purified as in lanes 7 and 8 or as expressed in vivo by pS637 inlane 5. This experiment demonstrates specific, high-levelphosphorylation of intact, recombinant human β-casein in E. coli K-12 ina bacterial system using a single construct.

Example 5

Production of β-casein in E. coli K-12: Phosphorylation ofextracellularly localized recombinant β-casein: Construct containing E.coli leader sequence, promoter, β-casein encoding sequence, pET-11d-CKIIβα

In this example we disclose the construction of a single plasmid that isused to transform E. coli K-12 and mediate production of extracellularlylocalized phosphorylated β-casein. To create a single construct designedfor secretion of phosphorylated protein to the periplasmic space of abacterial cell, the β-casein encoding sequence is put into an expressionvector containing a leader sequence that directs protein transport tothe periplasm. A polymerase chain reaction (PCR) is performed using theclone resulting from these procedures as the target DNA. The followingprimers synthesized at Midland Certified Reagent Co. (Midland, Tex.).can be used in the PCR, RO-4: 5', TGT AAA ACG GCC ACT-3' (Seq. ID No: 3)and RO-29: 5'-GGG GAT CCG TAC GCG TGA AAC-3' (Seq. ID No: 4) The baseunderlined in RO-29 incorporates a single base change to create an MluIsite at the end of the β-casein encoding sequence in order to eliminatethe bacterial initiation codon, methionine, for protein synthesis. Thisis done so that the resulting protein will have an amino acid sequenceidentical to that of human β-casein. The PCR fragment is then purified.The 3' end of the encoding sequence, which is not modified, is cut withBamH I. This fragment, containing a 5' blunt end and 3' BamH I end, iscloned in the expression vector pET-26b (Novagen, Madison, Wis.), whichcontains a T7 promoter, and cut at the blunt end with MscI and with BamHI. The construct described here contains the T7 promoter, but otherpromoter sequences could be used. The CKII βα encoding sequence isinserted as described above for pRjB-9. Expression is induced andWestern blot analysis is performed according to the procedures describedin Example 4.

A Western blot is performed to identify a protein, isolated from theperiplasmic space of the bacterial cells, that cross-reacts withantibody to phosphoserine and migrates similarly to native β-casein.This experiment demonstrates phosphorylation of recombinant humanβ-casein encoded by a sequence fused to a heterologous translationalstart and signal sequence, this sequence being preceded by a promotersequence, and the sequence to be phosphorylated being located in aplasmid containing a kinase encoding sequence such as CKII βα.Production of extracellularly localized phosphorylated protein has notbeen previously disclosed either in a one-vector or a two-vector system.

The advantage of extracellular over intracellular localization of theproduced phosphorylated protein lies in the ease of its purification.The periplasmic space of bacterial cells contains less extraneous matterthan the interior of the cell so that isolation of the purified proteinis expedited. This is particularly advantageous during commercialproduction.

Example 6

Comparison of Anti-Adhesion Bioactivity of Native and Recombinant Humanβ-Casein

Haemophilus are small, gram-negative bacilli with alipopolysaccharide-protein cell wall and are obligate parasites presenton the mucous membranes of humans and animal species. The surface ofmany but not all strains of Haemophilus influenzae is covered with apolysaccharide capsule. Nonencapsulated, nontypeable H. influenzaestrains colonize the upper respiratory tract in most individuals withinthe first few months of life and is the species most commonly associatedwith several diseases including otitis media and sinusitis (Murray etal., Medical Microbiology, 2d ed., p.260, 1994). They can alsoexacerbate chronic bronchitis.

An assay was performed to compare the activity of native human β-caseinwith recombinant human β-casein synthesized in cells containing pRJB-9in inhibiting adherence of H. influenzae to human pharynx cells.Comparisons were made between proteins phosphorylated with 0 to 5phosphates.

Cells and bacterial strains

Detroit 562 human pharynx carcinoma cells (DT 562) were obtained fromthe American Type Culture Collection (Rockville, Md.). The H. influenzaenontypeable bacterial strain was obtained from Dr. Lauren Bakaletz atthe Ohio State University.

Cell culture

DT 562 cells seeded into 96-well plates (Costar, Cambridge, Mass.) at adensity of 20,000-25,000 cells per well were cultured in Dulbeccos'sModified Eagle Medium (GIBCO, Grand Island, N.Y.) supplemented with 10%fetal bovine serum (FBS) (Hyclone, Logan, Utah). Cells were incubated ina humidified atmosphere of 95% air and 5% carbon dioxide at 37° C.Experiments were conducted when cells were at least 90% confluent.Plates containing cells were washed three times with 200 μL of HanksBalanced Salt Solution (HBSS) (GIBCO) to remove serum proteins beforethe addition of bacteria.

Native human β-casein

β-casein isolated from human milk was purchased from Symbicom AB, P.O.Box 1451, S-902 24 Umea, Sweden.

Separation of phosphoforms

Cells were harvested by centrifugation at 7000 ×g for 10 minutes at 40°C. Supernatant was removed and the pelleted cells were subjected to thefreeze/thaw method described in Johnson et al. (Bio/Technology, Dec. 12,1994, pp.1357-1360) to release the recombinant β-casein. Afterfiltration through a 0.45μ membrane, samples containing β-casein wereloaded onto an anion exchange column (Mono Q 10/10, Pharmacia Biotech,Uppsala, Sweden). Various phosphoforms were resolved on a lineargradient of 0 to 0.5M NaCl in 20 mM ethanolamine, 6M urea, at pH 9.5over a period of 50 minutes.

Different phosphoforms of recombinant β-casein were identified bycomparison of their elution times with those of purified native humanmilk β-casein.

Radiolabeling of bacteria

H. influenzae were streaked onto chocolate agar plates from frozenaliquots of a low passage number and incubated at 37° C. in a humidifiedatmosphere of 95% air and 5% carbon dioxide for 18 hours to obtain logphase cultures. Bacteria harvested in phosphate buffered saline (PBS)supplemented with 0.05% bovine serum albumin ((BSA) were centrifuged andresuspended in a volume of PBS/BSA yielding an optical density of 2.4 ata wave length of 600 nm (OD₆₀₀). ¹¹¹ Indium-oxine (¹¹¹ In) (Amersham,Arlington Heights, Ill.) was used to radiolabel the bacteria. 50 μCi ofthe ¹¹¹ In solution was added to 2.5 ml of the bacterial suspension andincubated for 20 minutes at 37° C. The radiolabeled bacteria were washedtwice with 10 ml HBSS to removed unbound ¹¹¹ In and resusupended in 5 mlHBSS supplemented with 30 nM HEPES buffer(N-2-hydroxyethylpeperazine-N'-2-ethane sulfonic acid). 25 μL of the ¹¹¹In labeled bacterial suspension were preincubated with 25 μL of the testagent in a polypropylene 96-well plate for 15 minutes at 37° C. to allowbinding of the agent to the bacteria.

Quantitation of adhesion

25 μl of the preincubation mixture containing radiolabeled bacteria andeither native human or recombinant β-casein was pipetted into each wellof the assay plate containing DT 562 cells. The assay plate wasincubated for 20 minutes at 37° C. to allow adhesion of the bacteria tothe cell monolayer. Nonadhering bacteria were removed by washing theplate three times with HBSS. The assay was terminated by the addition of100 μL of 0.05N sodium hydroxide to disrupt the cell monolayer and theadhering H. influenzae. The contents of each well was placed in a Cobrapolypropylene tube and counted on a Cobra gamma counter (Packard,Meriden, Conn.). Results were calculated by averaging the results offour replicates. Results are presented as the percent inhibition ofbacterial adhesion with native human or recombinant (pRJB-9) β-casein at6 different phosphorylation levels when compared to bacterial attachmentin control wells containing no test agent.

Results

Anti-adhesion activity is only seen consistently when β-casein isphosphorylated with 3, 4, or 5 phosphate groups. At lower levels ofphosphorylation little or no anti-adhesion was observed with eithernative or recombinant β-casein. However, at higher phosphoforms whenβ-casein had 3, 4, or 5 phosphates there was essentially no differencebetween the anti-adhesion bioactivity of native or recombinant (pRJB-g)human β-caseins. These results show that the bioactivity of β-casein ininhibiting adhesion of H. influenzae to human pharyngeal cells dependsupon the level of phosphorylation. Unphosphorylated or minimallyphosphorylated β-casein is ineffective. Attachment of 3, 4, or 5phosphate groups is required for inhibition of adhesion of H. influenzeto human pharyngeal cells. Results also demonstrate that phosphorylatedrecombinant β-casein made with the plasmid of the invention is aseffective as native human β-casein in inhibiting adhesion of H.influenzae. These results are summarized in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    ANTI-ADHESION BIOACTIVITY OF NATIVE AND                                       RECOMBINANT (pRJB-9)HUMAN BETACASEINS                                         Native Hβ    Recombinant Hβ                                               Test              Test                                                        Concentration                                                                        Adhesion   Concentration                                                                        Adhesion                                       Phosphoform                                                                         (mg/ml)                                                                              Inhibition                                                                         Phosphoform                                                                         (mg/ml)                                                                              Inhibition                                     __________________________________________________________________________    0P    1.00   15%  0P    0.40   -2%                                            1P    1.00    0%  1P    0.76   -4%                                            2P    1.00   -11% 2P    0.76   35%                                            3P    1.00   47%  3P    0.76   43%                                            4P    1.00   52%  4P    0.76   51%                                            5P    1.00   50%  5P    0.76   48%                                            __________________________________________________________________________

H. influenzae has been identified as a causative factor for otitis media(Murray et al., 1994). Since it has been demonstrated in the experimentsdescribed above that recombinant human β-casein phosphorylated in atleast three sites under the direction of the plasmid of the inventioninhibits adhesion of H. influenzae to human cells, it is concluded thatphosphorylated recombinant human β-casein, as described above, may beused in the prevention and treatment of otitis media in humans,particularly in human infants.

Therapeutic effects may be provided by enterally feeding or ingesting anenteral liquid nutritional product, such as infant formula, comprising atherapeutically effective amount of the phosphorylated recombinant humanβ-casein with 3 or more phosphate groups disclosed herein. Theattachment of H. influenzae to human oropharyngeal cells may also beinhibited by administering via a nasal passageway, or as a throat spray,a formulation containing a therapeutically effective amount ofphosphorylated recombinant human β-casein. Such a nasally administeredformulation may be in the form of either drops or a spray.Administration of enteral, throat spray and nasal products is believedto be effective because the interaction of human β-casein is believed tooccur by direct contact in the nasopharynx rather than after ingestionand digestion of the β-casein.

This invention will allow commercial-scale production of phosphorylated,recombinant mammalian proteins in microorganisms. The method of theinvention can be used to produce recombinant exogenous proteins,including but not limited to, recombinant human β-casein, in largequantities. Phosphorylation of β-casein in a bioreactor makes possiblelarge-scale synthesis in a fermentor of recombinant β-casein that isequivalent to native human β-casein. This will facilitate the productionof infant formula containing human β-casein in its native phosphorylatedstate. The method of the invention can also be used for phosphorylationof cell proteins, including receptors which are regulated byphosphorylation and dephosphorylation and thereby act as signals in cellmetabolism. The invention provides a cost-effective method ofphosphorylating peptide receptors and will be useful in the manufactureof pharmaceutical drugs.

The single plasmid system is preferable to a two-plasmid system forindustrial production of fermented proteins such as recombinant,phosphorylated human β-casein. Large-scale production of recombinantprotein without the selective pressure provided by antibiotics in thegrowth medium results in plasmid loss during the fermentation processsince the cells containing the plasmids would have no selectiveadvantage over those that contained only one or no plasmids, but wouldbe burdened by the presence of the plasmids resulting in slower growth.However, use of multiple antibiotics to provide the selective pressurenecessary to maintain both plasmids in the bacteria during fermentationfrequently retards bacterial growth and results in lower yield of thedesired recombinant product. Therefore, for industrial purposes thesingle-plasmid system disclosed herein is greatly preferable topreviously disclosed two-plasmid systems.

Phosphorylated recombinant human β-casein with 3 to 5 phosphate groupscan be incorporated into any standard or specialized enteral liquidnutritional product including but not limited to infant formulascontaining protein from non-human mammalian milk such as bovine or goatmilk or protein from vegetable sources such as soybeans or rice, as wellas other beverages consumed by young children. A product incorporatingphosphorylated recombinant human β-casein having 3 to 5 phosphate groupshas utility for the inhibition of attachment of H. influenzae to humancells and the treatment and prevention of otitis media in human infants.

The discovery disclosed herein of a novel method for producingrecombinant, phosphorylated human β-casein, with characteristics similaror identical to that of native human β-casein, makes feasible theaddition of this protein to infant formula so as to render it moresimilar to human milk with consequential benefits to developing infants.The disclosure of a method for producing recombinant, modified humanproteins in a bacterial system also makes feasible the addition of thehuman proteins to other food and pharmaceutical products.

Although specific preferred embodiments of the invention have beendescribed above with reference to the accompanying experiments anddrawings, it will be apparent that the invention is not limited to thoseprecise embodiments and that many modifications and variations could beeffected by one skilled in the art without departing from the spirit orscope of the invention as defined in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      CGCTGCAGCATATGCGTGAAACCATCGAATC31                                             (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base pairs                                                     (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      CGGGATCCTGGTCCTCGTGTTTAACTTTTTCAACTTTCTGTTTGTATTCGGTGATCGATTC61               (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      TGTAAAACGACGGCCAGT18                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      GGGGATCCGTACGCGTGAAAC21                                                       __________________________________________________________________________

What is claimed is:
 1. A plasmid comprising i) a promoter operablylinked to ii) a nucleotide sequence encoding a human protein, whereinsaid nucleotide sequence encoding said protein is regulated by saidpromoter, said nucleotide sequence being followed by iii) a nucleotidesequence encoding a kinase which specifically phosphorylates said humanprotein, wherein said nucleotide sequence encoding said kinase isregulated by said promoter.
 2. The plasmid of claim 1, wherein saidpromoter is an inducible promoter selected from the group consisting ofT7, λP_(L), λP_(R), and Tac.
 3. The plasmid of claim 1, wherein saidpromoter is a constitutive promoter selected from the group consistingof bla and spa.
 4. The plasmid of claim 1, wherein said human protein ishuman β-casein.
 5. The plasmid of claim 1, wherein said kinase is humancasein kinase (CKII).
 6. A plasmid comprising i) an inducible T7promoter operably linked to ii) a nucleotide sequence encoding humanβ-casein, wherein said nucleotide sequence encoding said human β-caseinis regulated by said promoter, said nucleotide sequence being followedby iii) a nucleotide sequence encoding CKII which specificallyphosphorylates said human β-casein, wherein said nucleotide sequenceencoding said CKII is regulated by said promoter.